脐血浆在CIK细胞培养中的应用

目的观察脐血浆体外培养脐血细胞因子诱导的杀伤细胞（CIK细胞）的增殖情况,探讨其替代AB血浆的可行性。方法无菌采集脐血并分离血浆,密度梯度离心法分离脐血单个核细胞,用脐血浆培养脐血CIK细胞,并与人AB血浆进行对照。计数培养不同时间CIK细胞数,流式细胞仪进行表型分析,以K562细胞为靶细胞,CCK-8法计算杀伤活性。结果脐血浆体外培养脐血CIK21d后,CD3^＋CD56^＋细胞扩增倍数、表达率、对白血病K562细胞的杀伤率与人AB血浆相比无显著性差异（P〉0．05）。结论脐血浆可以代替AB血浆进行脐血CIK培养。     

IL－2／抗IL－2抗体复合物体外诱导扩增外周血CD56^＋淋巴细胞的实验研究

目的探索一种在体外从人外周血单个核细胞（PBMC）扩增CD56^＋淋巴细胞的方法。方法以干细胞培养基（SCGM）为基础培养基,设计IL-2／抗IL-2抗体（IL-2／抗IL-2抗体质量比为1／20）实验组以及IL-2＋IL-15、相应因子浓度的IL-2和抗IL-2抗体三个对照组,从PBMC中诱导扩增CD56^＋淋巴细胞,流式细胞仪检测第7、10天CD3／CD56表达率,四甲基偶氮唑蓝法检测CD56^＋细胞对肿瘤细胞株K562的杀伤活性。结果培养第7、10天时,CD56^＋细胞分别扩增了（24．67±3．14）和（31．63±5．01）倍,其中CD3^＋CD56^＋和CD3^-CD56^＋细胞分别扩增（110．93±19．10）、（7．8±1．17）和（157．60±46．31）、（12．03±1．64）倍,实验组与对照组比较扩增倍数差别有统计学意义（P〈0．05）。培养第10天,效／靶比10：1时,实验组CD56^＋细胞对K562细胞的杀伤率为（83．46±1．56）％,与对照组比较具有更强细胞毒性作用（P〈0．05）。结论在SCGM为基础培养基条件下,IL-2／抗IL-2抗体复合物能更有效地扩增CD56^＋细胞毒性免疫效应细胞,为肿瘤过继免疫治疗提供了一种扩增外周血CD56^＋淋巴细胞的新方法。     

食管癌引流淋巴结细胞体外培养及其抑瘤作用的观察

目的观察食管癌引流淋巴结（TDLN）细胞体外培养结果及其抑瘤作用。方法术中切取食管癌患者的TDLN进行培养,分为3组：A组,培养基中添加IL-2;B组,添加IL-2＋IL-4＋粒—巨噬细胞集落刺激因子（GM-CSF）;C组,添加IL-2＋IL-4＋GM-CSF＋自身食管癌细胞抗原。于培养第1、7、14、21天行细胞计数;采用流式细胞技术测定TDLN细胞的CD3＋、CD4＋、CD8＋、CD5＋6、CD8＋3;MTT法测定各组TDLN细胞和淋巴因子激活杀伤（LAK）细胞对自体瘤细胞的抑瘤率。结果每1.0 g淋巴结组织培养第7、14、21天收获细胞数三组间差异不显著;A组第14天细胞明显多于第7天和第21天（P〈0.01）,第7天和第21天之间无明显差异;B组、C组结果类似。三组间CD4＋、CD8＋、CD8＋3细胞数相比,P〈0.05。未经培养的TDLN细胞、LAK细胞和各培养组TDLN细胞对自体肿瘤细胞的杀伤率有显著差异（P〈0.01）。结论TDLN细胞通过培养可以获得大量成熟树突状细胞和T细胞;培养基中添加GM-CSF和IL-4的TDLN培养后对自身肿瘤细胞的杀伤率最高。     

脐带血DCs对CIK细胞杀伤活性的影响研究

目的：探讨相同来源脐带血DCs对CIK细胞杀伤活性的影响作用。方法：采用相同来源的脐带血单个核细胞分别制备树突状细胞，CIK细胞，用流式细胞术检测免疫表型，MTT法检测CIK和CIK＋DC对K562，HL－60两种肿瘤细胞株的杀伤活性。结果：脐带血DC高表达CD1a,HLA-DR,CD80,CD86等抗原，CIK细胞是以CD3^ T细胞为主的异质性细胞群体，随培养时间延长CD3^ CD56^ 细胞比例明显升高，DC不改变CIK的免疫表型，但能明显促进其对K562和HL－60的杀伤活性（P＜0．05），结论：适量DC能增强CIK的杀伤活性，为DC的免疫治疗提供了新的思路。     

树突状细胞瘤苗对老年非小细胞肺癌自体癌细胞的杀伤作用

目的研究老年非小细胞肺癌（NSCLC）患者的树突状细胞（dendritic cell,DC）瘤苗对细胞因子诱导的杀伤细胞（cytokines-induced killers,CIK）的促生长作用,及CIK在体外对患者自身肿瘤细胞的杀伤作用。方法提取老年NSCLC患者外周血单个核细胞（peripheral blood mononuclear cells,PBMC）,分别培养成DC与CIK;将患者自身的肿瘤组织分离,培养出肿瘤细胞,制备肿瘤细胞冻融物作为抗原,用于负载DC,制备成Ag-DC,同时以单纯DC作为对照,各组DC经OK-432激活后,流式细胞仪检测DC表面分子的表达;将DC与CIK共培养,制备成DC-CIK、Ag-DC-CIK,同时以单纯CIK作为对照,四氮唑盐（MTT）法测定各组CIK对自身肿瘤细胞的杀伤力。结果负载肿瘤细胞冻融抗原的Ag-DC其表面CD1a、CD80、CD86、CD83、人类主要组织相容性抗原（HLA-DR）的表达上调,明显高于未负载肿瘤抗原的DC（P〈0.05）。各组DC与CIK共培养后,Ag-DC-CIK的增殖速度明显加快,其中CD3＋CD56＋细胞的含量增加,对自身肿瘤细胞的杀伤力亦明显加强,其作用均强于其他各组DC-CIK及CIK（P〈0.05）。结论老年NSCLC患者PBMC来源的DC在负载自身肿瘤细胞冻融抗原后,可分化为成熟DC,该DC可促进CIK的增殖,提高CIK对自身肿瘤细胞的杀伤力。     

树突状细胞增强细胞因子诱导的杀伤细胞抗神经胶质瘤细胞作用及机制研究

目的探讨细胞因子诱导的杀伤细胞（CIK）与树突状细胞（DC）共培养后DC-CIK混合细胞抗神经胶质瘤细胞的免疫作用。方法分离健康人外周血单个核细胞,分别于体外诱导DC和CIK,然后共培养成DC-CIK细胞。实验分DC组、DC-CIK组、DC-T组和CIK组。Elisa试剂盒检测各组培养上清中IL-12和IFN．1的含量,流式细胞仪检测细胞表型,CCK一8法体外检测对神经胶质瘤细胞的杀伤活性。结果DC-CIK组培养上清中IL-12和IFN-1的含量分别为（110．24±2．22）mg／L和INF·Y／（913．46±20．64）mg／L,明显高于其它三组（P〈0．05）。DC—CIK组cDi细胞（61．34-1．31）％、CD3／CD56细胞（29．4±1．03）％也明显增加（P〈0．05）。对神经胶质瘤细胞的杀伤活性,DC—CIK组为（54．67±2．62）％,与DC组（19．44±1．07）％、DC—T组（21．27±1．85）％和CIK组（36．52±2．06）％比较,差异有统计学意义（P〈0．05）。结论DC—CIK细胞能诱导明显的神经胶质瘤细胞杀伤活性,为颅内肿瘤的免疫治疗提供了依据。     

脐血DC与CIK共培养对白血病K562细胞杀伤活性的影响

目的观察细胞因子诱导的杀伤细胞（CIK）与同源树突状细胞（DC）共培养后DC—CIK细胞的增殖活性、表型的变化及其对白血病K562细胞杀伤活性的影响。方法采集健康产妇分娩正常足月胎儿脐血50ml,密度梯度离心法分离出脐血单个核细胞培养。收集非贴壁细胞用于诱导培养CIK,贴壁细胞诱导分化出成熟DC;将成熟DC和CIK按1：5的比例混合培养3d,用MTT法检测Dc—CIK共培养细胞对白血病K562细胞杀伤活性。结果DC与CIK共培养后,DC—CIK细胞群的增殖活性和杀伤活性明显高于单纯CIK。结论DC—CIK共培养可明显提高CIK增殖活性和细胞毒作用。     

DC、CIK白体回输对化疗后胃癌患者T淋巴细胞亚群及NK细胞的影响

目的研究树突状细胞（DC）与细胞因子诱导的杀伤细胞（CIK）自体回输对化疗后胃癌患者T淋巴细胞亚群及NK细胞的影响。方法将92例胃癌患者随机分为观察组及对照组各46例,两组均行常规化疗,观察组于化疗结束后1个月实施DC、CIK自体回输治疗,治疗时间〉2个疗程。采集两组化疗前后及观察组DC和CIK自体回输之后4周的外周血,以流式细胞仪对外周血T淋巴细胞亚群及NK细胞进行检测。结果两组化疗后CD;、CD4＋、CD4＋／cDf、NK水平均明显降低,观察组DC与CIK回输后CD^＋3、CD^＋4、CD^＋4／co^＋8、NK水平明显高于本组及对照组化疗后,P均〈0．05。结论化疗后胃癌患者DC、CIK白体回输后CD^＋3、CD4^＋4、CD^＋4／CD^＋8、NK水平升高,即细胞免疫功能得到改善。     

不同时期卡介苗免疫治疗对结核病大鼠疗效和CD4^＋CD8^＋双阳（DP）T细胞的影响

目的探讨不同时期给予卡介苗（BCG）免疫治疗对结核病化疗效果和T细胞严群的影响。方法80只PPD皮试阴性sD大鼠随机分成（1）化疗开始同步加BCG治疗组；（2）化疗1月后加BCG治疗组；（3）单纯化疗对照组；（4）模型效果判断组。各组于实验开始接受结核杆菌（Mtb）攻毒；第1个月末处死模型效果判断组全部大鼠作病理学和结核杆菌细菌学检查，其他各组于第1个月末接受抗结核化疗，其中第1组化疗开始加用BCG治疗，第2组化疗1个月后加用BCG治疗。观察各组大鼠死亡率；检测各组大鼠于Mtb攻毒前和Mtb攻毒后1、2、3、5个月末的外周血中CD3^＋、CD4^＋、CD8^＋、DPT细胞含量，第2、5个月末加查淋巴细胞绝对计数。结果（1）Mtb感染后1个月内1、3、4组各死亡2只大鼠，第2组死亡1只大鼠。化疗开始后4个月治疗期死亡率：化疗一开始即同步加BCG治疗组22．2％（4／18）高于化疗1月后加BCG治疗组（0／19），P〈0．05；化疗对照组为5．6％（1／18），（2）结核病鼠表现为CD4／CD8比值（可伴CIM^＋细胞）降低、CD8^＋（可伴DP）细胞升高，P〈0．05或P〈0．01；这种改变可在化疗后1～2个月内恢复到接近正常水平。（3）化疗开始同步加用BCG组治疗1个月时表现为T淋巴细胞（CD3^＋）、CD4^＋T细胞百分率显著降低，而淋巴细胞绝对计数和CD8^＋、DPT细胞百分率升高，P〈0．05或P〈0．01；治疗2个月后CD3^＋、CD4^＋T细胞恢复到正常范围而仍保持CD8^＋、DPT细胞较高水平。（4）化疗1个月后再加用BCG组在加用BCG后表现为CD3^＋、CD4^＋细胞百分率不降低而CD8^＋、DP细胞升高。结论淋巴细胞总数显著升高而T细胞占总淋巴细胞百分率显著降低是化疗开始同步加BCG治疗组第一个月死亡率升高的一个重要免疫学特征。DP细胞是抗结核保护性免疫相关细胞。BCG作结核病的免疫治疗应在化疗1个月后开始。     

支气管哮喘大鼠CD4^＋CD25^＋T细胞的变化及地塞米松干预作用的研究

目的研究支气管哮喘（简称哮喘）大鼠模型支气管肺泡灌洗液（BALF）、血液、脾脏CD4^＋CD25^＋T细胞的变化,及地塞米松对CD4^＋CD25^＋T细胞的影响。方法50只SD大鼠随机分为5组,空白对照（A）组,哮喘（B）组,地塞米松1（C）组、地塞米松2（D）组,地塞米松3（E）组。A组第1天给予腹腔注射生理盐水1ml,第15～21天每天给予生理盐水雾化。B、C、D、E组用卵蛋白建立哮喘大鼠模型,第1天,每只大鼠腹腔注射抗原1ml（卵蛋白1mg＋灭活百日咳杆菌9×10。个＋氢氧化铝干粉100mg）混悬液,第15～21天给予1％的卵蛋白雾化30min,C、D、E组于雾化后分别给予腹腔注射地塞米松0．2mg／kg、1mg／kg、2mg／kg。采用流式细胞仪检测的方法,观察大鼠体内BALF、外周血、脾脏CD4^＋CD25^＋T细胞的变化及使用不同剂量地塞米松后对其的影响。结果B组BALF、外周血、脾脏CD4^＋CD25^＋T细胞表达占CD4^＋T细胞的百分比分别是（42．21±5．62）％、（12．69±2．70）％、（11．15±1．05）％,A组结果分别是（18．76±5．85）％、（6．21±1．73）％、（7．85±2．13）％。B组与A组比较,差异均具有统计学意义（P〈0．01,P〈0．01,P〈0．05）;C组、D组、E组BALF中CD4^＋CD25^＋T细胞占CD4^＋T细胞的百分比表达分别是（10．49±4．03）oA、（13．28±5．12）％、（7．51±5．39）％,显著低于A组和B组,（P〈0．05,P〈0．01）;外周血中,C组（6．03±1．43）％、D组（4．88±0．95）％与A组（6．21±1．73）％比较,差异无统计学意义,E组（3．49士0．62）％与C组、A组比较,差异有统计学意义（P〈0．05）。脾脏中,c组（7．25±1．82）%、D组（8．63±3．18）％与A组（7．85±2．13）％比较,差异无统计学意义,E组（3．38±1．37）％与C组、D组、A组比较,差异有统计学意义（P〈0．05）。结论CD4^＋CD25^＋T细胞在哮喘大鼠体内有明显的优势表达,可能是哮喘发病的机制之一。地塞米松可以抑制CD4^＋CD25^＋T细胞的表达。BALF内CD4^＋CD25^＋T细胞的变化与外周血和脾脏的变化具有一致性,监测外周血或脾脏CD4^＋CD25^＋T细胞变化可了解肺部情况。     

Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.     

Enhancement of the anti-leukemic activity of cytokine induced killer cells with an anti-CD19 chimeric receptor delivering a 4-1BB-zeta activating signal

OBJECTIVE: There is growing interest in the use of cytokine-induced killer (CIK) cells in cancer therapy. In this study, we sought to maximize the antileukemic activity of anti-CD19 receptor-modified CIK cells against B-lineage acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: CIK cells were transduced with retroviral vectors carrying different types of anti-CD19 chimeric receptors: anti-CD19-zeta, anti-CD19-DAP10, anti-CD19-4-1BB-zeta, and anti-CD19-CD28-zeta. A truncated form of the receptor was used as a control. Transduced CIK cells were then analyzed for their cytotoxic activity against ALL cells and for their capability to proliferate and to release cytokines after ALL encounter. RESULTS: CIK cells were efficiently transduced with all the anti-CD19 retroviral vectors. Anti-CD19 receptor expression conferred powerful killing activity against ALL cells. However, there were clear advantages when receptors containing the co-stimulatory molecules 4-1BB or CD28 were transduced. Such cells had significantly more potent cytotoxicity than cells expressing the anti-CD19-zeta or anti-CD19-DAP10. Moreover, the presence of 4-1BB or CD28 in the receptor increased the production of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, TNF-beta, IL-5, IL-6, and IL-8 elicited by coculture with ALL cells. Notably, anti-CD19-4-1BB-zeta CIK cells secreted particularly low levels of interleukin-10 and proliferated strongly after contact with ALL cells. CONCLUSIONS: Anti-CD19 chimeric receptors delivering primary and costimulatory signals render CIK cells powerfully cytotoxic against ALL cells and induce secretion of immunostimulatory cytokines and proliferation. These results support the testing of genetically modified CIK cells in clinical trials.     

Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy

OBJECTIVE: Cytokine-induced killer (CIK) cells are ex vivo expanded cells enriched in CD3(+)CD56(+) natural killer T (NKT) cells with major histocompatibility-unrestricted cytotoxicity against several tumoral targets, except B-lineage acute lymphoblastic leukemia (B-ALL). We redirected CIK cells cytotoxicity toward B-ALL with a chimeric receptor specific for the CD19 antigen and then explored if modified-CIK cells maintain the same chemotactic properties of freshly isolated NKT cells, whose trafficking machinery reflects their preferential localization into the sites of B-ALL infiltration. MATERIAL AND METHODS: CIK cells were expanded ex vivo for 21 days and analyzed for expression of adhesion molecules and chemokine receptors regulating adhesion and homing toward leukemia-infiltrated tissues. CIK cells were then transduced with the anti-CD19-zeta-internal ribosomal entry site-green fluorescent protein retroviral vector and characterized for their cytotoxicity against B-ALL cells in a (51)Cr-release assay and for their trafficking properties, including chemotactic activity, adhesion and transendothelial migration, and metalloproteases-dependent invasion of Matrigel. RESULTS: Similarly to freshly isolated NKT cells, CD49d and CD11a were highly expressed on CIK cells. Moreover, CIK cells expressed CXCR4, CCR6, and CCR7 (mean expression 72%, 60%, and 32%, respectively), presenting chemotactic activity toward their respective ligands. Anti-CD19 chimeric receptor-modified CIK cells became cytotoxic against B-ALL cells (mean lysis, 60%) and showed, after exposure to a CXCL12 gradient, high capacity to adhere and transmigrate through endothelial cells and to invade Matrigel. CONCLUSION: The potential capacity to localize into leukemia-infiltrated tissues of anti-CD19 chimeric receptor-redirected CIK cells, together with their ability to efficiently kill B-ALL cells, suggests that modified-CIK cells represent a valuable tool for leukemia immunotherapy.     

Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3(+)CD56(+) CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.     

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402／R

ABM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2 000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhlFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.     

顺铂结合CIK细胞对裸鼠人胃癌模型体内的抗肿瘤作用

目的:探讨顺铂、CIK细胞以及顺铂联合CIK对裸鼠人胃癌移植瘤生长的抑制作用,为临床上联合应用顺铂和CIK细胞治疗胃癌提供实验依据.方法:应用淋巴细胞分离液分离外周血单个核细胞,给予多种细胞因子(rhIFN-g、CD3mcAb、rh1L-1、rhlL-2),诱导生成CIK细胞;培养人胃癌细胞株SGC-7901,接种至80只裸鼠右腋皮下,10 d后取移植瘤直径基本一致的裸鼠随机分4组:NS对照组、顺铂组、CIK细胞组和顺铂+CIK细胞组,每组16只.连续5 d在接种肿瘤细胞部位处给予相应注射治疗,观察其对胃癌移植瘤模型的抗肿瘤疗效.结果:与NS对照组相比,顺铂组、CIK细胞组、顺铂+CIK细胞组裸鼠人胃癌移植瘤模型治疗后肿块质量均减轻,生存期均明显延长,尤以顺铂+CIK组效果显著(P<0.01).裸鼠体内实验表明,顺铂+CIK细胞联合应用能够显著抑制胃癌细胞的生长,其抑瘤率可达57.8%,明显高于NS对照组(P<0.01).免疫功能检测显示红细胞C3b反应受体明显升高,红细胞免疫复合物受体明显降低(P<0.01).结论:顺铂联合CIK细胞对人胃癌移植瘤生长的抑制作用大于单独应用顺铂或CIK细胞.     

CIK细胞输注联合TACE治疗延缓原发性肝癌患者门静脉癌栓的形成

目的 探讨细胞因子诱导的杀伤细胞（CIK）输注联合经动脉插管化疗栓塞术（TACE）治疗原发性肝癌患者延缓门静脉癌栓形成的可能性。方法 选择2010年1月～2014年12月我院收治的原发性肝癌患者44例,22例接受TACE联合CIK细胞输注治疗,另22例只接受TACE治疗。采用Ficoll两步分离法分离外周血单个核细胞,经IFN-γ、抗CD3单克隆抗体和 IL-2诱导,使用流式细胞仪检测细胞CD抗原表达鉴定CIK细胞培养成功,常规行TACE治疗,在TACE治疗半月后输注CIK细胞悬液3次,细胞总数为1.0×109。治疗前后行腹部CT检查,确定门静脉栓子形成情况。结果 治疗后1 m、3 m和6 m,联合组患者门静脉癌栓发生率分别为4.5%、13.6%和27.2%,而TACE组则分别为9.0%、45.5%和63.6%（P<0.05）;在治疗3月时,联合组肿瘤大小为（4.77±1.27）cm,显著低于TACE组[（5.54±1.14）cm,P＜0.05];在治疗3月时,联合组血清甲胎蛋白水平为(83.14±31.91) ng/ml,也显著低于TACE组[(139.24±98.76) ng/ml,P＜0.05];两组肺部、颅脑和骨骼等远处转移发生率无显著性差异（P>0.05）。结论 CIK细胞输注联合TACE可明显减缓原发性肝癌患者门静脉栓子形成的时间,改善肝功能,同时能有效控制肿瘤的生长及扩散。     

Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma

AIM: To investigate the influence of autologous cytokine-induced killer (CIK) cells on the phenotypes of CIK effector cells, peripheral T lymphocyte subsets and dendritic cell subsets in patients with primary hepatocellular carcinoma (HCC). METHODS: Peripheral blood mononuclear cells (PBMC) were collected by a blood cell separator from 13 patients with HCC, then expanded by priming them with interferon-gamma (IFN-gamma) followed by monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2) the next day. The phenotypic patterns of CIK cells were characterized by flow cytometry on d 0, 4, 7, 10, 13 and 15 of incubation, respectively. Then, 5 mL of venous blood was obtained from HCC patients before or 8-10 d after CIK cells were transfused into patients to assess the influence of CIK cells on the percentages of effector cells, and proportions of DC1 or DC2 in peripheral blood by flow cytometry. RESULTS: After two weeks of in vitro incubation, the percentages of CD3(+)CD8(+), CD3(+)CD56(+), and CD25(+) cells increased significantly from 33.5+/-10.1%, 7.7+/-2.8%, and 12.3+/-4.5% to 36.6+/-9.0% (P<0.05), 18.9+/-6.9% (P<0.01), and 16.4+/-5.9% (P<0.05), respectively. However, the percentages of CD3(+)CD4(+) and NK cells had no significant difference. The percentages of CD3(+) and CD3(+)CD8(+) cells were kept at high levels during the whole incubation period, but those of CD25(+), and CD3(+)CD56(+) cells began to decrease on d 7 and 13, respectively. The proportions of type I dendritic cell (DC1) and type II dendritic cell (DC2) subsets increased from 0.59+/-0.23% and 0.26+/-0.12% before CIK cell therapy to 0.85+/-0.27% and 0.43+/-0.19% (all P<0.01) after CIK cell transfusion, respectively. The symptoms and characteristics of HCC patients were relieved without major side effects. CONCLUSION: Our results indicated that autologous CIK cells can efficiently improve the immunological status in HCC patients, and may provide a potent approach for HCC patients as the adoptive immunotherapy.     

Antitumor activities of human autologous cytokine—induced killer（CIK）cells against hepatocellular carcinoma cells in vitro and in vivo

AIM：To characterize the anticancer function of cytokine induced killer cells(CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma(HCC),we evaluated the proliferation rate phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo.METHODS:Peipheral bolld mononuclear cells(PBMC) form healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma(IFN-γ),interleukin-1(IL-1),IL-2,and monoclonal antibody(mAb) against CD3.The phenotype and characterization of CIK cells were identified by folw cytometric analysis.The cytotoxicity of CIK cells was detemined by ^51Cr release assay.RESULTS:The CIK cells were shown to be a heterogeneous population with different cellular phenotypes.The percentage of CD3^+/CD56^+ positive cells,the dominant effector cells,in total CIK cells from healthy donors and HCC patients,significantly increased form 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation,which suggested that the CD^3+ CD56^+positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study.After 28 day in vitro incubation,the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number respectively,CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer(LAK) cells and PBMC cells,In in vivo animal experiment.CIK cells had stonger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells(mean inhibitory rate 84.7%VS52.8%,P&lt;0.05) or PBMC(mean inhibitory rate 84.7%VS 37.1%,P&lt;0.01).CONCLUSION:Autologous CIK cells are of highly efficient cytotoxic effcetor cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patrents.     

化疗联合自体细胞因子诱导杀伤细胞治疗急性白血病的临床观察

目的评价化疗联合自体细胞因子诱导的杀伤细胞(CIK)治疗急性白血病(AL)的疗效。方法选择经化疗达完全缓解(CR)>6个月的AL患者41例。19例患者接受化疗联合自体CIK细胞治疗,22例接受同期单纯化疗作为对照组。CIK细胞治疗采用血细胞分离机大量采集患者的外周血单个核细胞,用抗CD3单抗、IL2、IFNγ培养10d,将细胞洗涤后经静脉于化疗后第一天回输给患者。结果CIK组19例患者化疗同时共接受52疗程CIK细胞治疗,每例患者平均接受2~3个疗程CIK细胞治疗。每疗程回输CIK细胞总数为(22~300)×109/L,平均(142±85)×109/L。4年持续CR(CCR)率CIK组为734%;单纯化疗组4年预期CCR率为273%。两者差异有统计学意义(P<0005)。接受≥3个疗程CIK治疗的10例患者至观察截止时均处于CCR,中位CCR期43个月(23~52个月);接受<3个疗程CIK治疗的患者4/9例复发。结论化疗联合自体CIK细胞治疗AL的CCR率明显优于单纯化疗;疗效与疗程有关,疗程≥3个患者的疗效优于疗程<3个的患者。